The present invention relates generally to methods of preparing and using a separation medium for purifying IgG. In particular, the present invention relates to the preparation and use of Protein A and variants of Protein A, including recombinantly produced variants, to purify IgG by coupling the Protein A or variant thereof to a base matrix with a bridging group.
Adsorbents which exhibit IgG-binding proteins have been used to capture IgG in aqueous media for more than twenty years. Initially, native Protein A (GB 1,441,979 (Sjxc3x6qvist)) was used. Later recombinantly produced forms of Protein A and were developed (WO 8400773 to Lofdahl, et al.; EP 262,192 to Guss, et al.; and U.S. Pat. No. 5,082,773 to Fahnestock).
Protein A has a broad IgG-specificity with respect to animal species, but the specificity may vary with respect to subclasses (for instance, human IgG3 will not bind to Protein A). Protein G binds to all IgG subclasses of a majority of important mammalian species. The advantage of Protein A compared to Protein G is that the binding of IgG is weaker, and consequently milder conditions can be used to release IgG from Protein A. This is of importance for the purification of individual monoclonal antibodies.
Recombinant techniques enable simple mapping of IgG-binding proteins with regard to the functionality of different domains. In the case of Protein A, it was found that the native form contained five consecutively ordered IgG-binding C domains (E, D, A, B and C), followed by an X-domain which did not bind IgG. The new technique facilitated the preparation of IgG-binding fragments and variants where one or more amino acids was/were replaced, added or removed. Unless otherwise indicated, reference to Protein A indicates the native form, or IgG-binding fragments and variants of Protein A that have the same IgG specificity as native Protein A. Variants of Protein A which contain cysteine were produced relatively early on, and the inserted cysteine residue was used for binding to base matrices. It was considered important not to place cysteine as a C-terminal residue. A variant having cysteine as the penultimate amino acid in the C-terminal part was bonded to activated Thiol SEPHAROSE(copyright) (Pharmacia Biotech AB, Uppsala, Sweden) via disulfide bond formation and studied as an IgG-separation medium (T. Profy (Repligen); EP 284,368 and U.S. Pat. No. 5,084,559). Similar studies were also presented in FASEB 87, Mar. 29-Apr. 2, 1987 (Poster N44, Profy, et al). The results obtained with three other variants (1, 2 and 5 domains) of Protein A with cysteine in a C-terminal linker sequence (amino acid 10 from the C-terminal) (Ljungquist, et al, Eur. J. Biochem. 186 (1989) 557-561) were later presented. These latter variants were also coupled covalently via disulfide bound formation to thiopropyl SEPHAROSE(copyright). Immobilization to tresyl chloride or tosyl chloride activated gels was suggested as an alternative, with the intention of avoiding reductively sensitive linking groups. An equimolar relationship was found between IgG binding capacity and the number of domains for one-domain and two-domain variants. The five-domain variant never bonded more than the double molar amount of IgG. IgG-capacities comparable to those achieved earlier with soluble forms of native Protein A were obtained it was later found that in certain applications, non-cys-containing variants can give molar binding ratios which lie between two and three).
Parallel herewith, Genex (U.S. Pat. No. 4,977,247 (Fahnestock, et al) has produced a recombinant variant of rProtein G-cys in which cysteine is located at the C-terminal end of an IgG-binding domain. In preparing separation media based on this Protein G variant, the choice was to bind rprotein G-cys covalently to aminohexyl-agarose activated with the bi-functional reagent N-sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate (U.S. Pat. No. 4,977 247, Claim 1 and column 18, lines 22-37). GAMMABINDG(copyright) Plus (Pharmacia Biotech AB) is a commercially available solid phase rProtein G-cys product with cysteine as the C-terminal residue. The product is synthesized by coupling the cysteinyl residue to aminohexyl agarose activated with N-sulfosuccinimidyl 4-(N-maleimidomethyl) cyklohexane-1-carboxylate.
As far as we are aware, the variant of the matrix-bound rProtein A-cys produced by Repligen has not found favor commercially. The reason may be that the coupling to the matrix is through unstable structures (xe2x80x94Sxe2x80x94Sxe2x80x94), although the reason may also be due to factors unknown to us. Whatever the reason, however, the adsorbent that totally dominates commercially makes use of native Protein A or different forms of recombinant Protein A that lack cysteine. The market for products based on Protein G has been substantially smaller, probably because Protein A has more advantageous binding properties.
The object of the invention can be summarized in the desire to provide adsorption media which have a) the IgG-binding specificity of Protein A; b) at least the same stability as other adsorbents based on native Protein A; c) the same or improved capacity to bind IgG. compared to earlier known variants of matrix bound rProtein A-cys (primarily calculated as the ratio mol IgG per mol cys-variant of Protein A with one, two or more IgG binding domains). For variants with two or more domains this means molar ratios xe2x89xa72. Fulfillment of these objects will enable more effective processes to be used for purifying IgG from different starting materials.
The main aspect of the invention is a separation medium which comprises a base matrix substituted with groups of the formula I:
xe2x80x94Bxe2x80x94Xxe2x80x94rProtein A-cys,xe2x80x83xe2x80x83I
where
a. rProtein A-cys is recombinantly produced Protein A which contains cysteine in its amino acid sequence;
b. B is a bridge which binds to the base matrix; and
c. X contains a heteroatom N or S originating from rProtein A-cys.
The characteristic feature is that X is a thioether sulphur (xe2x80x94Sxe2x80x94) and/or a secondary amine (xe2x80x94NHxe2x80x94); i.e. in one and the same separation medium X may be either or both a thioether sulphur and/or a secondary amine, with preference for greater than 50%, such as essentially 100%, of all X being thioether sulphur.
The optimal molar ratio between the total IgG binding capacity and the amount of Protein A on the matrix may vary depending on the number of IgG-binding domains that are present in the Protein A of the adsorbent. For single-domain variants the ratio is 1 and for 2-domain variants the ratio is ≈2. For three-, four- and five-domain variants the ratio is ≈2 or preferably  greater than 2. The maximum value is determined by the number of IgG-binding domains and is therefore contingent on the particular Protein A construction used.
In principle, B can be anything that has satisfactory stability under the conditions applied in the adsorption/desorption of IgG (time, temperature, pH, etc.). Examples of relevant structures in the bridge xe2x80x94Bxe2x80x94 are amide, ester, ether, thioether, hydrocarbon chains, azo, carbamate, etc. Hydrocarbon chains present in xe2x80x94Bxe2x80x94 may be straight, branched or cyclic, and normally have only saturated carbon atoms (2-10 carbon atoms, preferably 2, 3 or 4 carbon atoms to retain a pronounced hydrophilic nature). It is preferred that bridge xe2x80x94Bxe2x80x94 binds to the base matrix via an ether structure or an amide/ester structure. It is also preferred that B comprises a straight, branched or cyclic saturated hydrocarbon chain which may optionally compromise at one or more positions in the hydrocarbon chain an inserted oxygen or nitrogen, or at one or more positions in the hydrocarbon chain a hydrogen substituted with an amino or a hydroxy group. For stability reasons one and the same carbon atom should bind at most one oxygen or nitrogen atom. The structures that are preferred in xe2x80x94Bxe2x80x94 are those which occur when rProtein A-cys is coupled to the matrix via an epoxy group or epihalo; i.e. xe2x80x94Bxe2x80x94 includes at the end nearest X, the structure
xe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2xe2x80x94
X becomes a secondary amine or thioether, depending on whether the xcex5-amino group in a lysine, an N-terminal amino group, or a thiol group in cysteine (preferably located at/or near the C-terminal end) is coupled.
The aforesaid bridge structures can be formed in accordance with current techniques, for instance by the use of bi-functional coupling reagents, such as epichlorohydrin, bisepoxide (such as 1,4-bis (2,3-epoxypropoxy) butane, N-sulfosuccinimidyl 4-(N-maleimidomethyl)-cyklohexane-1-carboxylate, etc. Relevant base matrices may be activated with such reagents, so that they will contain groups that react more or less selectively with thiol or amino groups. Preferred coupling reagents and conditions give very little coupling at primary amino groups (xcex5-amino in lysyl and N-amino terminal groups).
Relevant forms of rProtein A-cys have an amino acid in the native sequence replaced with cysteine. Alternatively, cysteine can be present in an amino acid sequence (linker) which has been fused to a terminal, or as an insert in the native sequence or an IgG-binding part thereof. Cysteine may also be included in a peptide linker that preferably is N-terminal or C-terminal to an IgG-binding domain. Generally speaking, a terminal cysteine is preferred to an internal cysteine. The length of the linker used is normally not critical and may vary from one to fifty amino acid residues, for instance. For all cysteine modifications it is imperative that the IgG binding ability is not lost or reduced substantially.
rProtein A-cys may also, be modified in other ways. For example, rProtein A-cys may be a fusion protein which, in addition to featuring an IgG-binding domain from Protein A, also includes one or more IgG-binding domains from Protein G or from some other IgG-binding protein (c.f. Guss, et.al, EP 262, 192). The native domains maybe permutated, occur one or more times, or some may be deleted. Native non-IgG-binding domains may be missing totally or in part.
rProtein A-cys can be prepared in accordance with current techniques (Profy T, EP 294,386; and Ljungquist, et al, Eur. J. Biochem. 186 (1989), 557-561).
The base matrix is a hydrophilic polymer which contains a plurality of amino groups and/or hydroxy groups, primarily the latter. The base matrix is normally insoluble in aqueous media. The base matrix may originate from a polysaccharide, such as dextran, cellulose, starch, agarose, pullulan, xylan, etc., which may be cross-linked and/or provided with different groups suitable for the use intended. Among synthetic polymers can be mentioned polymers of hydroxyalkyl acrylates or corresponding methacrylates, polyvinyl alcohols, polymers of vinyl hydroxyalkyl ethers, etc. To the extent that a polymer is soluble, it can be made insoluble, for instance crosslinked or adsorbed or covalently bound to a support which is insoluble in aqueous media, for instance a styrene divinyl benzene copolymer. The base matrix can also be in the form of particles that may be more or less spherical and/or porous or non-porous. One particular type of matrix is porous hydrophobic particles made of divinyl benzene-styrene copolymer or some other hydrophobic polymer/copolymer, the inner and/or outer surfaces of which have been hydrophilized and provided with OH-groups. In a preferred embodiment, the matrix is insoluble in aqueous media, porous and based on a polysaccharide.
Another main aspect of the invention involves binding (adsorbing) IgG to a separation medium. IgG is then contacted with a separation medium in accordance with the aforegoing. Adsorption normally takes place from an aqueous solution derived from serum or a cell culture capable of producing IgG. Suitable conditions lie in the range 0-35xc2x0 C., pH 6-8, salt concentration 0.1-3 M (depending on the type of IgG to be bound). Before desorption of bound IgG, the separation media are normally washed, suitably with a buffer essentially with the same pH as the adsorption buffer, whereafter desorption is effected conventionally, for instance by treatment with a buffer which has a pH beneath 5. The conditions should be non-denaturing.
Binding of IgG to the separation medium has a broad field of use. It can be utilized in processes involving capture of IgG from a solution, i.e. to separate IgG dissolved in an aqueous solution from other components present therein. Binding of IgG may be a part-step in a chromatography process or in a batch-wise process. Binding of IgG may also be a part of a so-called immunoassay or an extracorporeal process for removing IgG from whole blood or plasma. The primary area of use is found in purifying IgG (including monoclonal IgG antibodies).
A very expedient embodiment of the invention is to couple rProtein A-cys by a C-terminal cysteine to a chromatographic particulate matrix containing densifying filler particles, such as ANVAL(copyright) (Anval, Torshxc3xa4lla, Sweden). The so obtained chromatographic support has been found very useful for chromatographic separations of IgG in stabilised fluidised beds. See our contemporary patent application SE 9503926-9 relating to xe2x80x9cAdsorption Method and Separation Mediumxe2x80x9d. With regard to chromatography on expanded/fluidized beds, reference is made to WO 9218237 (Pharmacia Biotech AB).